Composite Separators For Blood Collection Tubes

ABSTRACT

Sample collection tubes and methods of producing the same are provided. Contemplated collection tubes comprise a tube having a separator substance disposed therein. In some aspects, the separator substance preferably maintains a predetermined flowability during irradiation or heat sterilization, and can subsequently polymerize upon exposure to a UV light or other suitable source. In other aspects, the separator substance preferably includes a soft gel component (thixotropic gel), and a photocurable sealant component that is formulated to polymerize and form a solid barrier between fractions of a liquid.

This application claims priority to European Patent Application serialnumber 14190680.0, filed Oct. 28, 2014. This and all other extrinsicreferences are incorporated herein by reference in their entirety. Wherea definition or use of a term in an incorporated reference isinconsistent or contrary to the definition of that term provided herein,the definition of that term provided herein applies and the definitionof that term in the reference does not apply.

FIELD OF THE INVENTION

The field of the invention is sample separation technologies.

BACKGROUND

Analysis of blood samples often requires separation of whole blood intotwo or more fractions, for example, a serum fraction and acell-containing fraction. It is well known in the art that whole bloodseparation can be carried out through centrifugation by disposing wholeblood into a blood collection tube, placing the tube into a centrifuge,and spinning down the blood.

Unfortunately, once the blood separates, the fractions of the wholeblood can remix causing contamination of the fractions throughdiffusion, agitation, sample extraction, or other undesirableinteraction. Ideally, the two fractions should remain isolated to ensureno contamination occurs when accessing the desired fraction.

In an attempt to overcome the problems discussed above, efforts havebeen put forth in providing separator gels disposed in a bottom portionof a tube. These separator gels are intended to help preserve analytestability, to decrease manual labor (pipetting to a secondary tube), andallow for delayed processing that may result from the need to transportspecimens from draw locations to testing facilities. Some functional andperformance properties of the gels typically include the following: (1)the gel properties prevent flow before use to eliminate reflux potentialduring blood draw; (2) the gel has a density value between cells andserum/plasma (about 1.04 specific gravity); (3) the gel is thixotropic(shear thins in centrifuge under ordinary clinical lab protocols); (4)the gel liquefies and flows past blood cells and proteins duringcentrifugation; and (5) the liquefied gel re-gels at a layer betweencells and serum after centrifugation and adheres to the tube wall.Furthermore, the components of the gel generally should not interferewith blood components or assays used in the clinical lab.

Unfortunately, known separator gels suffer from one or more direct andindirect disadvantages, including for example: interference (certainassays are known to be problematic); analyte drift due to permeability,cell trapping in or on the surface of the gel; physical contamination ofanalyzer probes or with electrophoresis gels; inaccuracies caused byre-spinning; the need for aliquotting (with attendant manual labor costsand the potential for relabeling errors); incomplete aspiration whichnegatively affects standardization and results in wastage; andstorage/shipping issues such as gel dislodging, freeze-thaw issues, andthe inability to remix a sample with a soft gel barrier.

In an attempt to overcome some of the problems associated with separatorgels, effort has been put forth in attempting to provide compositionsand methods for whole blood separation that ensures the separatedfactions of whole blood are effectively protected against contaminationdue to undesirable sample interactions. For example, Applicant hasobtained several patents for previous efforts directed towardsphotopolymer serum separators and methods (e.g., U.S. Pat. Nos.7,674,388, 7,673,758, 7,775,962, 7,971,730, 7,780,861, 8,206,638,8,282,540). Photopolymer serum separators can be advantageous inproviding a solid interface (when the photopolymer gel is polymerized)between cells and serum or plasma, which allows for complete aspirationof the sample. Additionally, a tube comprising some photopolymerseparator gels (before polymerization to form a solid) can be shipped,refrigerated or frozen with repeated freeze-thaw cycles. Still further,the tube can be mixed without disrupting the barrier to ensure uniformsampling of the test fraction (e.g., blood is not remixed when tubes areagitated), the tube is free of soft gel material (after polymerization)that can clog analyzer probes or pipette tips, or come into contact withtest fraction to interfere with susceptible laboratory assay methods(e.g., electrophoresis).

Unfortunately, some known separator compositions and methods have beenproblematic for various reasons, including the high cost of photocurablecompositions, the need to formulate a thixotropic composition, the heatproduced in exothermic polymerization reactions which may affectanalytes, inability to sterilize via irradiation while maintainingflowability of the separator compositions for subsequent UV curing(e.g., after shipment of the sterilized composition in sample collectiontubes), and volume-dependent UV-light exposure requirements.

Thus, there is still a need for improved separation technologies.

SUMMARY

The present inventive subject matter provides apparatus, compositions,systems and methods that generally attempt to solve the problemsdescribed above by providing a sample collection tube comprising: (i) atube having a lumen, and (ii) a composite separator substance disposedwithin the lumen, wherein the separator substance comprises a gelcomponent layer material and a photocurable sealant component layermaterial. More specifically, the gel component layer material couldcomprise a thixotropic separator gel that is formulated to become aliquid when stirred or shaken, and the photocurable sealant componentlayer material could comprise a photopolymer sealant. The gel componentlayer material and the photocurable sealant component layer materialcould be separate layers (e.g., before irradiation sterilization, beforecentrifugation, before curing, after irradiation sterilization, aftercentrifugation, after curing, before and after irradiationsterilization, before and after centrifugation, before and aftercuring). Additionally or alternatively, the gel component layer materialand the photocurable sealant component layer material could comprise amixture.

The photocurable sealant component could optionally be anti-thixotropic.Additionally or alternatively, the photocurable sealant component couldbe formulated to polymerize within ten minutes to a hardness of at least1 on the Shore 00 hardness scale when triggered by a suitable energysource (e.g., UV light (arc lamps, microwave power bulbs, LEDs)).

There are numerous factors that can affect the curing of thephotocurable sealants and photocurable separator substances of theinventive subject matter. The suitable light source could emit a lighthaving an intensity of between 5-100 W/cm², between 10-75 W/cm², between15-50 W/cm², or any other suitable intensity—all measured at a distanceof 10 cm from the light source. Additionally or alternatively, thesuitable energy source could produce a light having a maximum peak at awavelength of between 50-400 nm, for example between 200-280 nm (UVC),between 280-315 nm (UVB), between 315-400 nm (UVA), or between 200-400nm. Additionally or alternatively, the suitable energy source could emita light with a peak irradiance of between 0.1-10 W/cm², for example,between 0.3-1 W/cm², between 1.5-2.5 W/cm², or between 0.5-3.5 W/cm².Additionally or alternatively, the light produced by the suitable lightsource could arrive at the surface to be cured with a radiant energydensity of between 0.3-8 J/cm², for example, between 1-5 J/cm², orbetween 1-2 J/cm².

Viewed from another perspective, the photocurable sealant componentcould comprise a promoter to allow polymerization within ten minutes toat least 1 on the Shore 00 hardness scale when triggered by a suitableenergy source. Additionally or alternatively, the photocurable sealantcomponent could be formulated to polymerize within ten minutes to atleast 10 on at least one of the Shore A hardness scale and the Shore Dhardness scale when triggered by a suitable energy source. Viewed fromyet another perspective, it is contemplated that the photocurablesealant component, after polymerization triggered by a suitable energysource, could be a solid with respect to a probe.

The present inventive subject matter also provides apparatus, systemsand methods in which a collection tube includes a separator substancethat could maintain analyte levels (e.g., potassium levels and glucoselevels) within acceptable thresholds for extended periods of time. Inone embodiment, potassium levels are stable within 10% of an initiallevel before centrifugation and glucose levels are stable within 5%.Viewed from another perspective, one or both of the gel component andthe photocurable sealant component (e.g., the entire separatorsubstance) could be formulated to maintain a potassium level of a sampledisposed in the tube within 25%, within 15%, within 10%, or even within5% of an initial potassium level for at least four days. It is alsocontemplated that one or both of the gel component and the photocurablesealant component could be formulated to maintain a glucose level of asample disposed in the tube within 25%, within 15%, within 10%, or evenwithin 5% of an initial potassium level for at least four days, morepreferably for at least five days.

Additionally or alternatively, the separator substance couldadvantageously be biocompatible with whole blood, and formulated to havea density between an average density of a serum/plasma fraction of wholeblood and a cell-containing fraction of whole blood (e.g., about 1.04g/cm³). The photocurable sealant component typically has a density thatis slightly lower than the gel component such that, upon curing, thephotocurable sealant component sits on top of the gel component andprovides a clear barrier. Additionally or alternatively, the separatorsubstance could be flowable in whole blood before curing, andimmobilized after curing forming a solid layer sealant.

Viewed from another perspective, the inventive subject matter includesmethods of producing sample collection tubes. A contemplated step ofmethods described herein could include disposing a photocurable sealantcomponent layer material into a lumen of the tube. A further step couldinclude disposing a gel separator component layer material into thelumen of the tube. The aforementioned steps could be completed in anysuitable order such that the gel component could be disposed above orbelow the photocurable sealant component. In some methods, thephotocurable sealant component layer material is depositedbefore/beneath the gel component layer material, and is configured toform a solid seal layer above the gel component layer material uponcuring. In some contemplated methods, the gel component is first placedin a tube, followed by a photocurable sealant component. Uponcentrifugation, the sealant component could rise above the gel componentto form a seal layer that acts as a barrier between the gel componentand a fraction of plasma, serum, or other sample. It should beappreciated that the gel component and the photocurable sealantcomponent composes a composite separator substance of the inventivesubject matter as described above.

Some contemplated methods could additionally comprise disposing a sample(e.g., blood) into the lumen of the tube, and centrifuging the samplecollection tube with the composite separator substance and sampledisposed therein. Additionally or alternatively, the sample collectiontube could be exposed to a LUV light (e.g., during or aftercentrifugation) to solidify the photocurable sealant component.Additionally or alternatively, where whole blood is disposed in thetube, a method of the inventive subject matter could comprise separatinga cell-containing fraction of the whole blood from the serum fraction(e.g., by physically removing a fraction via a pipette) after exposingthe tube to UV light or other suitable energy source to initiatepolymerization of the photocurable sealant component.

Other optional steps of some contemplated methods include, among otherthings, sterilizing a collection tube before or after disposing acomposite separator substance therein, and introducing a vacuum into alumen of the tube to facilitate the draw of a predetermined volume ofliquid. The step of sterilizing could be performed in any suitablemanner, including for example, gamma irradiation, e-beam irradiation, orsterilizing the components by exposing to beat (e.g., to at least 250degrees Celsius). The step of introducing a vacuum could be performed inany suitable manner. For example, an evacuation-closure device could beused to at least partially evacuate the interior of the tube and apply astopper to the opening of the tube. Additionally or alternatively, avacuum could be introduced into the collection tube by decompressing thevolume of the lumen using any suitable pump.

It should be appreciated that a sample collection tube of the inventivesubject matter could be used for fractionation of any suitable sample,including for example, whole blood. Suitable separator substances areformulated to have a suitable density intermediate to the fractions ofthe sample being separated. Where the sample being separated is wholeblood, for example, the separator substance could be formulated to havea density between an average density of a serum fraction of whole bloodand a cell-containing fraction of whole blood, and to be flowable inwhole blood. Once the separator substance separates fractions of thesample being separated, the photocurable sealant component/layer can behardened through polymerization to prevent mixing of the separatedfractions.

Other components could advantageously be included in a tube of theinventive subject matter, including for example, an anticoagulant (e.g.,where a sample comprises plasma) or a clot activator (e.g., where asample comprises serum).

The inventive subject matter also provides apparatuses, compositions,systems and methods of providing polymerizable compositions that aresterilizable via irradiation or application of heat, while maintaining apredetermined flowability effective to allow sedimentation of thecomposition under a centrifugal force to a position between acell-depleted phase of whole blood and a cell-enriched phase of wholeblood. In some aspects, the polymerizable composition comprises anoligomer, a photoinitiator, a stabilizer and an antioxidant, and can bedisposed within a lumen of a sample collection tube. Where the tube andpolymerizable composition are sterilized via irradiation or heat, thepredetermined flowability of the composition is preferably maintained ina manner that allows for subsequent polymerization of the compositionvia UV curing.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawings in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates a tube with a gel separator, and a tube with acomposite separator before and after centrifugation and curing.

FIG. 2 illustrates a separator tube comprising a photopolymer separatorof the inventive subject matter.

DETAILED DESCRIPTION

The following discussion provides many example embodiments of theinventive subject matter. Although each embodiment represents a singlecombination of inventive elements, the inventive subject matter isconsidered to include all possible combinations of the disclosedelements. Thus if one embodiment comprises elements A, B, and C, and asecond embodiment comprises elements B and D, then the inventive subjectmatter is also considered to include other remaining combinations of A,B, C, or D, even if not explicitly disclosed.

Separator substances of the inventive subject matter are advantageousover existing separator substances at least for the following reasons:

-   -   Ability to separate any cell trappings that may occur within the        gel from the upper test fraction.    -   Reducing the UV light intensity and time exposure required for        completely curing (solidifying) the barrier, at least because        the volume of the photocurable sealant component required is        reduced. For example, we have reduced the time required (with        the same UV light intensity) from over a minute to between 10-20        seconds. This reduction has the additional benefits of        decreasing the effect of UV on light-sensitive pigments,        improving work flow by decreasing processing time, and        decreasing exothermic heat production during curing, which may        affect certain blood components such as enzymes.    -   Allows for complete aspiration of a sample without substantial,        if any, contamination from the gel component, at least because        the solidified photocurable sealant component acts as a barrier        from the gel component.    -   Allows for mixing and re-mixing of a cell-free fraction of whole        blood to ensure uniform sampling.    -   Prevents remixing of blood that can occur when soft gel        separators are physically disrupted by agitation, for example,        during shipping or mixing.    -   Allows for repeated freeze/thaw cycles of the tube. Freezing of        serum or plasma is common practice, for example, for future        testing or for compliance with certain regulatory requirements.        Where a gel separator is used to separate fractions of a sample,        the fraction below the separator will typically freeze before        the gel, thereby expanding and distorting the shape of the gel        separator barrier. Since a separator substance of the inventive        subject matter includes a photocurable sealant component that is        formulated to solidify upon exposure to a suitable energy        source, some or all of the problems typically associated with        freezing and thawing separator tubes are significantly reduced        or even eliminated.    -   The photocurable layer can be minimized in volume thereby        reducing cost.    -   The phocurable layer does not need to be thixotropic since        thixotropic soft gel is loaded into the tube above the        photocurable layer and no flow will result prior to use. This        allows for simple compositions with less cell trapping, and        eliminates the need for additives that may contribute to        degrading blood cells or interference with lab assays.    -   Decreases labor costs associated with removing serum or plasma        to a secondary tube.    -   Decreases potential for re-labeling errors associated with        removing serum or plasma to a secondary tube.

Feasibility has been demonstrated using a photocurable sealantcomprising: (1) at least one of a monomer, and an oligomer (e.g., acombination (e.g., Ebecryl, Cytec) thereof), with (2) a photoinitiator(e.g., Additol® BDK, Additol® TPO) and (3) a stabilizer (phenothiazine).It should be appreciated that any commercially suitable photocurablesealant component can be used. Suitable photocurable sealant componentsare typically at least one of a gel (e.g., when a gelling agent is added(e.g., DBS or silica)) and flowable (with whole blood) prior topolymerization, and can solidify when exposed to a suitable energysource (e.g., UV light). Examples of suitable photocurable sealants caninclude, among other things, MLA (e.g., M1L1A1), MLAI (e.g., M1L1A1 andphenothiazine), MAI (e.g., M1A1 and phenothiazine), LAI (e.g., L1A1 andphenothiazine), and LMA (e.g., L1M1A1). As used herein, M=a monomer(e.g., M1, which is a monomer Trimethylolpropane propoxylate triacrylatefrom Sigma-Aldrich Cat. No. 407577); L=an oligomer (e.g., L1=Ebecryl 230from Allnex, previously from Cytec Industries, Inc.); A=a photoinitiator(e.g., A1=Additol BDK); I=a stabilizer (e.g., phenothiazine). See Table1 below. LAI (e.g., L1, A1 and phenothiazine) can be included in someespecially preferred photocurable sealant components, as it can have thedesired density range of 1.00-1.09 g/cm³.

Other examples of suitable photocurable sealants include LAIR, LAIE(e.g., L1, A1, phenothiazine and tocopherol), and LAIER, wherein R=agelling agent (e.g., DBS, silica), and E=at least one of an antioxidantand a radical scavenger (e.g., Vitamin E, butylated hydroxytoluene(BHT), butylated hydroxyanisole (BHA), carotene, bilirubin, ascorbicacid). While R and E are generally not necessary, each can provideadvantageous features to the photocurable sealant. More specifically, agelling agent is generally not a necessary component of the photocurablesealant because a thixotropic soft gel can be loaded into the tube abovethe photocurable layer such that no flow will result prior to use.Nonetheless, it may be desirable to have a thixotropic sealant, forexample, where it is desirable to have the sealant disposed in the tubeon top of the soft gel component. Additionally, a radical scavenger suchas compounds having Vitamin E activity (e.g., tocopherol), while notnecessary, can allow the photocurable sealant to be sterilized viairradiation (e.g., gamma, e-beam) without curing (e.g., by changing thedensity properties), rather than requiring heat sterilization tomaintain a flowability effective to allow sedimentation between twofractions of a sample.

TABLE 1 ABBREVIATION NAME R Gelling Agent (e.g., DBS, silica) M MonomerM1 Trimethylolpropane propoxylate triacrylate L Oligomer L1 Ebecryl 230from Allnex A Photoinitiator A1 Additol BDK I Stabilizer I1Phenothiazine E Antioxidant/radical scavenger (e.g., Vitamin E, BHT,BHA, carotene, bilirubin, ascorbic acid) D Density adjuster (e.g.,Ebecryl 113 from Cytec)

It is contemplated that composite separators of the inventive subjectmatter, in one or both of the photocurable and gel components, couldinclude a polymer such as silicon oil, polyamides, olefinic polymers,polyacrylates, polyesters and copolymers thereof, polysilanes andpolyisoprenes. Additionally or alternatively, composite separators couldinclude a filler (e.g., silica, latex, other inert material) to adjust adensity of the separator or component thereof. Additionally oralternatively, composite separators could include additional materialsor reagents to achieve a desired purpose (e.g., EDTA (chelating agent),or heparin, citrate, dextrose (anticoagulants)).

Similarly, it should be appreciated that any suitable gel component canbe disposed in a tube of the inventive subject matter. Suitable gelcomponents include those that liquefy during centrifugation and re-gelthereafter, and can include, for example, off the shelf gels (e.g., BDVacutainer® SST™, BD Vacutainer® PST™, Vacuette® blood collection tubeswith gel separators, PPMA serum separator gel tubes, Polypropylene serumseparator gel tubes), or any other commercially suitable gel that isformulated to, upon centrifugation, be located between two fractions ofa sample (e.g., between a serum and a blood clot, between serum and cellcontaining fraction of whole blood).

FIG. 1 illustrates a control tube 100A and a tube of the inventivesubject matter 100B. Control tube 100A is a sample collection tube(e.g., Vacutainer) including a commercially available gel separator110A. Tube 100B is a sample collection tube including a commerciallyavailable gel separator 110B, and a photocurable sealant 120. A sampleof whole blood 125 is transferred into control tube 100A, and anothersample of whole blood 130 is transferred into control tube 100B. Uponcentrifugation, gel separator 110A (including some cells trapped fromthe cell-enriched fraction) is positioned between a denser fraction ofwhole blood 125A, and a less dense fraction of whole blood 125B in tube100A. Gel separator 110B (including some cells trapped from thecell-enriched fraction) is similarly positioned between denser and lessdense fractions of whole blood, 130A and 130B, respectively.

Advantageously the photo-sealant 120, which includes no gelling agentsor thixotropy-modifying constituents, is clear and free of cell trappingbefore and after each of centrifugation and UV curing in tube 100B. Celltrapping is undesirable because of analyte leach into the test fraction.Cells frequently adhere to the upper layer of soft gels such as 110A and110B as shown in FIG. 1. However, they can be separated from the plasmaby the photo-sealant 120.

The following data shows improved analyte stability using a compositeseparator of the inventive subject matter (photogel plasma separatortube) when compared to using a soft gel alone.

BD Plasma Separator Tube Photogel Plasma Separator Tube ComprehensiveImmediate Freeze/ Immediate Freeze/ Metabolic Panel Spin 24 Hours 5 DaysThaw Spin 24 Hours 5 Days Thaw sodium mEq/L 142 139 140 143 140 140 142143 potassium mEq/L 3.7 3.7 4.1 4.5 3.6 3.6 3.7 3.8 chlorides mEq/L 106104 105 108 106 105 107 108 CO2 mEq/L 29 27 26 22 27 25 25 22 glucosemg/dL 102 100 93 91 104 105 102 106 urea nitrogen mg/dL 16 15 16 16 1716 16 16 creatinine mg/dL 0.6 0.8 0.8 0.8 0.7 0.8 0.8 0.5 calcium mg/dL9.4 9.3 9.5 9.7 9.2 9.4 9.5 9.6 total protein g/dL 6.8 6.8 6.4 7 6.8 76.7 7 albumin g/dL 4 4 4 4 4 4.1 4.1 4 alkphos IU/L 37 39 36 38 36 38 3937 AST IU/L 17 15 19 26 17 17 18 16 ALT IU/L 15 16 19 16 16 17 17 13bilirubin mg/dL 0.6 0.7 0.6 0.5 0.5 0.6 0.6 0.4

Some sample collection tubes of the inventive subject matter cancomprise a tube, plug, and a separator substance having a gelcomponent/layer and a photocurable sealant component/layer disposed in alumen of the tube. The collection tube can be manufactured out of asuitably rigid material to support a vacuum within the lumen. Examplematerials include hard plastics, glass, or other similar materials. Thelumen is preferably of sufficient volume to hold a desirable sample ofwhole blood or other liquid. Typical volumes range from a few ml to 10ml or greater. The plug could fit sufficiently snug into the tube tomaintain the vacuum within lumen. An example of an acceptable tube thatcan be used to produce a collection tube of the inventive subject matterincludes the Vacutainer® specimen collection products developed byBecton, Dickenson and Company (Franklin Lakes, N.J. USA 07417).

The term “tube” is used euphemistically to refer to vessels having acavity. Although a preferred embodiment includes a tube, one shouldappreciate that other vessels having a cavity can also be used whilestill falling within the scope of the inventive subject matter. Forexample, it is contemplated that collection tube could be replaced withother vessels that can contain a liquid or optionally support a vacuum.Alternative examples of vessels include flasks, jars, beakers, bottles,blood collection bags, or phials. Vessels beyond mere tubes also haveutility when the inventive subject matter is applied to alternativemarkets beyond blood collection.

In a preferred embodiment, a collection tube is produced by disposing acomposite separator within a lumen of the tube, and introducing a vacuumwithin the lumen in preparation for sale. It can be preferred (e.g., forcost and cure time purposes) that no more than about 1 ml, or about 1gram, of the separator substance is disposed into the lumen for atypical 10 ml collection tube. Additionally or alternatively, it can bepreferred that no more than 50%, more preferably no more than 25%, andeven more preferably no more than 10% of the separator substancecomprises the photocurable sealant layer. It is contemplated that otheramounts of the separator substance (or layer thereof) could be used insome embodiments to fit a specific use case. For example a smallerversion of a tube could require less of a separator substance, while alarger version might require more to make an adequate sealed barrier.

In some embodiments, collection tubes are sterilized to satisfy theInternational Organization for Standardization (ISO) protocols beforebeing sold. For example, tubes can be sterilized (preferably withoutsubstantial polymerization of the separator substance or portionthereof) using gamma radiation (e.g., from a Cobalt source (e.g., Cobalt60)), using e-beam radiation (e.g., from an e-beam generator), gas(e.g., ethylene oxide), or a heat between 100 to 250 degrees Celsius oreven more. Viewed from another perspective, the separator substance canbe effective to allow irradiation, gas, or heat sterilization withoutcuring more than 40%, more preferably without curing more than 30%, andto allow subsequent polymerization via UV or other curing. An optionalvacuum can be introduced, for example, by simply decompressing thevolume of the tube's lumen by using a suitable pump.

All suitable sterilization times are contemplated (e.g., less than 10minutes, less than 5 minutes, less than 2 minutes, between 5-120seconds, between 5-90 seconds), where the collection tubes (andseparator substances) are e-beam sterilized at dosages of between 5-25kGy, more typically between 10-20 kGy. All suitable sterilization timesare contemplated (e.g., less than 10 minutes, less than 5 minutes, lessthan 2 minutes, between 5-120 seconds, between 5-90 seconds), where thecollection tubes (and separator substances) are gamma sterilized atdosages of between 5-25 kGy, more typically between 10-20 kGy. It hasbeen observed that with gamma sterilization, weaker sources with lowerdose delivery rates were more likely to cure the compound. The doserequired by the ISO depends on, among other things, the bioburden of theobject being sterilized. The radiation time required depends on not onlythe sterilization technique used, but also, for example, the bioburdenof the object being sterilized, and the radiation dose (kGy).

It is also contemplated that a collection tube could be sterilized, anda sterilized separator substance could be added to the tube.Additionally or alternatively, a user could add one or more separatorsubstances to a collection tube after purchase, as opposed to having aseparator substance pre-disposed within the tube.

Where a sample (e.g., whole blood) is added to a collection tube of theinventive subject matter, centrifugation could separate the whole bloodinto a serum fraction and a cell-containing fraction. When each layer(gel/photocurable sealant) of the separator substance has a density thatis intermediate to that of serum faction and cell-containing fraction,it can migrate between the two fractions during centrifugation, therebyisolating the fractions from each other. The separator substance canthen be rapidly hardened through polymerization when triggered by asuitable energy source to provide a solid barrier between the twofractions.

As discussed above, the suitable light source could emit a light havingan intensity of between 5-100 W/cm², between 10-75 W/cm², between 15-50W/cm², or any other suitable intensity. Additionally or alternatively,the suitable energy source could produce a light having a maximum peakat a wavelength of between 50-400 nm, for example between 200-280 nm(UVC), between 280-315 nm (UVB), between 315-400 nm (UVA), or between200-400 nm. Additionally or alternatively, the suitable energy sourcecould emit a light with a peak irradiance of between 0.1-10 W/cm², forexample, between 0.3-1 W/cm², between 1.5-2.5 W/cm², or between 0.5-3.5W/cm². Additionally or alternatively, the light produced by the suitablelight source could arrive at the surface to be cured with a radiantenergy density of between 0.3-8 J/cm², for example, between 1-5 J/cm²,or between 1-2 J/cm².

One exemplary suitable light source is a custom light box made byHeraeus that produces a light having a maximum peak at a wavelength of385 nm, and a peak irradiance of 2.2 W/cm². This light source was usedwith a power setting of 25% of maximum optical output power of 25 W.Some of the tested photocurable substances had a volume of between0.25-1 mL, was disposed in vacutainer tubes, and the suitable energysources were light emitting diodes emitting energy at between 380-390nm, with a peak irradiance of 2.2 W/cm². However, it should beappreciated that one or more factors of exposure (e.g., irradiance,wavelengths, radiant energy) can be modified, concentrations ofsubstance components could be modified (e.g., antioxidant concentration,photoinitiator concentration), or different energy sources could beused, to achieve a similar cure time for smaller or larger volumes.

In some aspects of the inventive subject matter, a separator tube can beprovided with (1) both a polymerizable composition and a thixotropicgel, as shown in FIG. 1, or (2) a polymerizable composition alone, asshown in FIG. 2. As illustrated, sample collection tube 200 comprises apolymerizable composition 210 of the inventive subject matter (e.g.,LAIE) disposed therein and cured after centrifugation to a positionbetween a cell-depleted phase 220 and a cell-enriched phase 230 of wholeblood.

The polymerizable composition preferably comprises at least three of thefollowing components: an oligomer (L) (e.g., an acrylate containingoligomer and a methacrylate containing oligomer), a photoinitiator (A),a stabilizer (I) and at least one of a radical scavenger and anantioxidant (E).

The polymerizable composition can advantageously have a compositioneffective to allow irradiation sterilization (e.g., gamma, e-beam)without loss of a predetermined flowability (e.g., effective to allowsedimentation of the composition under a centrifugal force to a positionbetween two phases of a sample (e.g., a cell-depleted phase and acell-enriched phase of whole blood)).

Viewed from another perspective, the composition can be effective toallow irradiation or heat sterilization without curing the compositionmore than 40%, more preferably without curing the composition more than30%, and to allow subsequent polymerization via UV or other curing.

The subsequent polymerization via UV curing could occur minutes (e.g.,more than 30 minutes), hours (e.g., more than 1 hour, more than 2 hours,more than 5 hours, more than 10 hours), days (more than 1 day, more than5 days, more than 10 days, more than 15 days, more than 20 days, morethan 25 days), months (more than 1 month, more than 2 months, more than6 months, more than 9 months) or even years (more than 1 year, more than2 years, more than 3 years, more than 5 years or even longer) afterirradiation sterilization occurs. In some embodiments, the polymerizablecomposition can be subject to a radiation dose of between 5 and 35 kGy,inclusive, more preferably a radiation dose of between 10 and 30 kGy,inclusive, and even more preferably a radiation dose of between 10 and20 kGy, inclusive, without loss of the flowability. Viewed from adifferent perspective, the polymerizable composition can be subject to aradiation dose of less than 30 kGy, more preferably less than 20 kGy,and even more preferably less than 17 kGy to both (1) allow irradiationsterilization without loss of the predetermined flowability, and (2)allow subsequent polymerization via UV curing.

It is contemplated that a photoinitiator (e.g., Azobisisobutyronitrile,Benzoyl peroxide, Camphorquinone, a phosphine oxide photoinitiator, aketone-based photoinitiator, a benzoin ether photoinitiator) could bepresent in the polymerizable composition in any suitable concentration.In some preferred embodiments, the photoinitiator is present in thecomposition in a concentration of less than 5 wt %, more preferably in aconcentration of less than 2 wt %, and even more preferably in aconcentration of less than 1.5 wt %. Additionally or alternatively, apolymerizable composition of the inventive subject matter could comprisea photoinhibitor.

It should be noted that a radical scavenger or antioxidant is notnecessary in all contemplated polymerizable compositions. However, whereincluded (e.g., to facilitate maintaining flowability throughirradiation sterilization via gamma beam or electron beam), it iscontemplated that the at least one of the radical scavenger and theantioxidant (e.g., tocopherol) can be present in the polymerizablecomposition in any suitable molar concentration. Applicant surprisinglyfound that where a radical scavenger such as tocopherol is included,some compositions of the inventive subject matter (e.g., LAIE) willmaintain flowability during irradiation sterilization at a radiationdosage of more than 3 kG, while some other compositions (e.g., LAI) willnot maintain flowability under the same radiation dosage. Viewed fromanother perspective, LAI was found to only maintain flowability duringirradiation sterilization up to a radiation dosage of approximately 3kG. In some preferred embodiments, the at least one of the radicalscavenger and the antioxidant comprises tocopherol and is present in thecomposition in a molar concentration of at least 75 mM, more preferablyat least 100 mM, and even more preferably at least 135 mM. Lowerconcentrations of tocopherol (e.g., less than about 75 mM) are notpreferable for various reasons. For example, separator substances withlower tocopherol concentrations can only maintain flowability at lowerradiation dosages, which may not allow for cost-effective sterilizationunder the ISO protocol. Additionally, separator substances with lowertocopherol concentrations typically require lower photoinitiatorconcentrations (e.g., less than 1 wt %), which generally requires alonger cure time.

Additionally or alternatively, the polymerizable composition could bepolymerizable by UV curing (e.g., after irradiation sterilization(gamma, e-beam)) to form any suitable polymer, including for example, anacrylate polymer, a methacrylate polymer, an epoxy polymer, apolyurethane, or a thiol-ene polymer.

Viewed from a different perspective, a polymerizable composition of theinventive subject matter could comprise one or more of a polymercontaining a terminal epoxy group, a polymer containing a pendant epoxygroup, an epoxy-siloxane resin, an epoxy-polyurethane, anepoxy-polyester, epichlorohydrin, a polyhydric diol, a polyhydricpolyol, a polymer comprising a terminal or pendant isocyanate group, apolymer comprising at least two hydroxyl groups, a polyhydric diol, apolyhydric polyol, an aliphatic monomeric polythiol, an aliphaticdithiol, an aromatic dithiol, a polymeric polythiol, an acrylate, amethacrylate, an alkenyl, and a cycloalkenyl. Where a polymerizablecomposition of the inventive subject matter is subject to a suitableenergy source (e.g., a UV energy source), it is contemplated that thecomposition can be cured to a hardness of at least 1 (e.g., a hardnessof at least 10) on a Shore A hardness scale over a period of less thanten minutes, more preferably a period of less than 5 minutes, morepreferably a period of less than 60 seconds, and even more preferably, aperiod of less than 20 seconds. Viewed from another perspective, it iscontemplated that the composition can be cured to a hardness of at least1 (e.g., a hardness of at least 10) on the Shore 00 hardness scalewithin at least 10 minutes, more preferably a period of less than 5minutes, more preferably a period of less than 60 seconds, and even morepreferably, a period of less than 20 seconds. Viewed from yet anotherperspective, it is contemplated that the composition can be cured to ahardness of at least 1 (e.g., a hardness of at least 10) on a Shore Dhardness scale over a period of less than ten minutes, more preferably aperiod of less than 5 minutes, more preferably a period of less than 60seconds, and even more preferably, a period of less than 20 seconds.

Experiments

Various candidate compositions variably comprising an oligomer or amonomer (or both), a photoinitator, a stabilizer, an antioxidant, adensity adjuster or gelling agent (or both) were tested to determine,among other things, whether a predetermined flowability could bemaintained during irradiation (e-beam or gamma) sterilization at variousradiation dosages and for various time periods. More specifically, thecompositions were tested with respect to the following:

-   -   a. Final Density—tested by pycnometry and performance in whole        blood during centrifugation. The final density of some preferred        compositions were between 1.04-1.08 g/cm³.    -   b. Interference with lab tests—tested by comparing results to        those obtained in blood collected in BD tubes. No interference        was inferred when the means of the measurements were within        assay CVs. The lab test comparison included the whole process of        using the separator in the centrifuge and curing with UV.        Monomers and oligomers were also tested with respect to leaving        the blood in contact with the cure photopolymer for up to 8 days        looking for delayed interference.        -   i. Comprehensive metabolic panel (sodium, potassium,            chloride, CO2, creatinine, bilirubin (direct and total),            total protein, AST, ALT, alkaline phosphatase, glucose, urea            nitrogen, albumin)        -   ii. Immunoassays (PSA, testosterone, estradiol, TSH,            thyroxine (free T4), ferritin, sensitive CRP)        -   iii. Electrophoresis (serum protein electrophoresis and            immunofixation (fraction quantitation (5 fractions),            paraprotein identification), lipid panels by            electrophoresis)        -   iv. Lipids (total cholesterol, LDL-c, HDL-c, VLDL-c, Lp(a),            triglycerides)        -   v. Molecular tests (DNA (braf), exosome RNA (HBB, ACTB,            DEFA3), GAPDH, ITGA2B))        -   vi. Therapeutic drug monitoring (amikacin, primadone,            lidocaine, caffeine, acetaminophen, NAP, procainamide)        -   vii. Platelet, red cell and white cell counts (with            differentials) in plasma, including platelet aggregation to            ristocetin, collagen, and ADP.    -   c. Less interference was observed with one or more of i-vii        above when the amount of photopolymer or gel separator was        reduced. The components of either have the potential for        interference. When a specific analyte is interfered with by the        gel component, that component can be reduced. When a specific        analyte is interfered with by the photopolymer component, that        component can be reduced.    -   d. Hemolysis—measured by index on automated analyzers, visual        assessment and elevate potassium levels.    -   e. Cure times with UV lamps (arc lamps, microwave power bulbs,        LEDs)        -   i. Increasing antioxidant concentration beyond (for example            over 500 mM tocopherol) adversely affects cure times            (prolongs). Increasing the photoinitiator concentration            beyond approximately 3% to offset this effect adversely            affected the function of the antioxidant in preserving the            compound through sterilizing irradiation.    -   f. Heat production when cured.    -   g. Shrinkage when cured.    -   h. Ability to be sterilized by heat or irradiation (ebeam and        gamma at various doses and dose delivery schema).        -   i. For heat sterilization, no antioxidants were required and            several combinations of L and M satisfy the other            performance requirements.        -   ii. For sterilization by irradiation, a given composition is            more likely to work if the dose is delivered quickly (for            example by ebeam rather than by gamma).            -   1. LAI can be sterilized with up to 3 kGy without an                antioxidant.

The monomers tested (some in combination for density adjustment)included M1, 1,6 hexanediol ethoxylate diacrylate, Poly(ethylene glycol)methyl ether methacrylate, Poly(ethylene glycol) diacrylate (Cat. No.437441), Poly(ethylene glycol) diacrylate (Cat. No. 475629),Trimethylolpropane propoxylate triacrylate, and 1,6-Hexanediolethoxylate diacrylate (Cat. No. 497134). Of the monomers tested, M1worked best. Unfortunately, compositions including one or more monomers(e.g., M1) resulted in shrinkage after curing and were therefore notconsidered optimal for use in sealant compositions. However, it iscontemplated that the compositions could be modified, for example, toaddress the cure rate and temperature produced to prevent or reduceshrinkage.

The oligomers tested were all in the Ebecryl series, and included L1-L10(various oligomers obtained from Cytec). Of the oligomers tested, L1,L2, L6, L8, L9 and L10 were better suited for maintaining flowabilitycompared to L3-L5 and L7. L1 performed the best with respect to, forexample, less heat generation during curing, less shrinkage (if any),density, viscosity, less bubble trapping, and less interferenceexhibited using standard serum tests, and the other oligomers wereabandoned for failing to meet the criteria. L8 and L9 resulted ingreater interference with enzymes than L1, while L10 resulted in visibleinteraction with cured materials. The plasma visibly dissected into thetop layer of the barrier.

Although L1 had the best overall performance of the oligomers tested inthe Ebecryl series, it should be appreciated that several oligomers wereable to maintain flowability during irradiation sterilization. It shouldalso be appreciated that adjustment, for example, to a radiation dosage,sterilization time, or concentration of a constituent of thephotocurable substances could be made by a person having ordinary skillin the art to allow various other oligomers to be included in aseparator substances formulated to maintain flowability duringirradiation sterilization.

The photoinitiators tested in 1 wt % concentration in M1, included(Azobisisobutylonitrile): 254 nm; Benzophenone: 254 nm;4-(Dimethylamino)benzophenone: 356 nm;4,4′-Bis(dimethylamino)benzophenone: 370 nm;4,4′-Bis(diethylamino)benzophenone: 379 nm; and A1: 365 nm. Thewavelength of photoinitator was chosen by transmission through PET tubesthat would be used. A1 was found to have superior performance, usingcriteria including, compatibility with blood, cure times, heatgeneration, and miscibility with oligomers and monomers. Some of theother photoinitiators could be used, for example, with a greater curetime or a different light source.

A1 was tested in various concentrations in L1 (between 0.5-8 wt %,inclusive). 1%±0.5% was found to be optimum where antioxidant(s) werepresent. However, the concentration of A1 could be increased up toapproximately 3% without adversely affecting the function of vitamin E.Additol TPO, which has a longer wavelength absorption to match selectedUV sources, was also tested in various concentrations in L1. WhenAdditol TPO was tested in 1% concentration with L1, I1 and vitamin E in200-300 mM concentration, the cure time was somewhat better than when A1was tested in 1% concentration with L1, I1 and vitamin E in 200-300 mMconcentration. Furthermore, no interference was seen using acomprehensive metabolic panel.

The stabilizer tested is Phenothiazine in 0.1 wt % concentration. Thisstabilizer was present in all samples. However, it is contemplated thatany suitable stabilizer at any suitable concentration could be used,including for example, suitable stabilizers that would work in anevacuated (low O₂) atmosphere.

The antioxidants tested included alpha-tocopherol—2 mM-500 mM, carotene,bilirubin, BHT, BHA, tempo, and 4-OH-tempo. While tocopherol, tempo and4-OH-tempo each maintained flowability during irradiation in dosagesabove 15 kGy (carotene, bilirubin, BHT and BHA failed), Tempo and4-OH-tempo caused hemolysis and lab interference. Tempo, however, wasable to maintain flowability during irradiation in dosages of up to 37kGy, and no heat step was required. Tocopherol was found to have thebest performance.

The density adjusters tested included Ebecryl 113 from CYTEC. A physicalgel formulation denoted LARID (D=Ebecryl 113 from Cytec) was tested andshown to have a good cure time and little or no interference with mostlab tests. LARID comprises:

-   -   a. 30% L=EBECRYL 230 and 70% D    -   b. wherein:        -   i. 1% (of L+D) A1 (Additol BDK);        -   ii. 8.19% (of L+D) R, fumed silica R1 (AEROSIL R805 from            Degussa); and        -   iii. 0.1% (of L+D) I (Phenothiazine from Sigma).

The LARID formulation was found to be non-sterilizable by irradiation(while maintaining flowability) because no antioxidant was present.

The gelling agents tested included fumed silica and DBS(1,3:2,4-dibenzylidene sorbitol) and cosolvent NMP(N-Methylpyrrolidone). DBS performed worse than silica in terms ofsterilization by irradiation while maintaining flowability. With 0.6%DBS and 1.8% NMP, the L1A1I1 system gels. It is shear thinning and has adensity lower than a BD gel. The UV curing time was about the same asL1A1I1R1 and the cured gel was able to survive one round with a liquidnitrogen-hot water test, which means the cured gel could survive manyrounds of freeze-thaw cycles.

EXAMPLES

The following experimental data is provided to exemplarily illustratevarious aspects of the inventive subject matter presented herein. Morespecifically, the data illustrates the surprising effects of tocopheroland Additol BDK at specified concentrations in maintaining flowabilityof a composition (to allow for sedimentation between two phases of wholeblood upon centrifugation) after irradiation sterilization at specifiedradiation dosages. As shown, compositions comprising an oligomer(EBECRYL 230), a photoinitiator, Additol BDK, in a concentration of lessthan 2 wt %, and a radical scavenger (tocopherol, in a concentration ofat least 75 mM) surprisingly maintained flowability after irradiationsterilization (gamma or e-beam) at a dosage of less than 20 kGy. Wherelower concentrations of tocopherol or photoinitiator were present,radiation protocols could be modified (e.g., to require a higher doserate (e-beam), post-sterilization heat, and lower dosages (kGy), longercure times). Where amounts lower than about 60 mM tocopherol werepresent, it was generally observed that the total dose deliverable waslower, making the sterilization protocols not feasible. In contrast,compositions lacking a radical scavenger and compositions having aphotoinitiator concentration of greater than 3 wt % (e.g., greater than5 wt %) were unable to maintain flowability after irradiationsterilization.

Photo- Pass Tocopherol initiator Post- (maintain Conc. Conc. DoseSterilization Sterilization flowability) Cure Composition (mM) (wt %)(kGy) Method Heat or Fail Time L1A1I1- 0 1 3-5 Gamma None Fail DBS(Failed (L1A1I1 = worse Ebecryl 230, with Additol increased BDK, DBS)phenothiazine) (DBS = dibenzylidene sorbitol) L1A1I1 0 1 17 e-beam NoneFail L1A1I1 0 1 15 Gamma None Fail L1A1I1 0 1 10 Gamma None Fail LARID140 1 16 e-beam None Fail LAIE 0.1-2.0 1 25 Gamma None Fail (Ebecryl230, Additol BDK, phenothiazine, tocopherol) LAIE 10, 20 1 25 Gamma NoneFail LAIE 75 .5 15 e-beam None Fail LAIE 75 1 15 e-beam 60-70 C. Fail 60min LAIE 75 .5 10 e-beam 60-70 C. Pass Typically 60 min <5 minutes LAIE100 1 12 e-beam 60-70 C. Pass Typically 60 min <1 minute LAIE 120 1 15e-beam 60-70 C. Pass Typically 60 min <1 minute LAIE 120 1 16.1 Gamma 70C. Fail 60 min LAIE 100, 120, 140 1 16.1 e-beam None Almost LAIE 140 1.512 e-beam 60-70 C. Pass Typically 60 min <1 minute LAIE 140 1 16.1e-beam 70 C. Pass Typically 60 min <1 minute LAIE 140-200 2-5 15-20e-beam None Fail or Gamma LAIE 140 2-3 17 e-beam None Fail LAIE 140 1 16e-beam None Close LAIE 140, 200 1 16 e-beam 60-70 C. Pass Typically 60min <1 minute LAIE 145 1 16 e-beam 50 C. Fail 2 hours LAIE 145 1 12e-beam 70 C. Pass Typically or 60 min <2 Gamma minutes LAIE 145 1 16e-beam 70 C. Pass Typically or 60 min <2 Gamma minutes LAIE 200 5 15-20e-beam None Fail or Gamma

As illustrated herein, a technical effect of some aspects of theinventive subject matter is that tocopherol included in a sealantcomposition in a suitable concentration range (e.g., between 75-200 mM,between 75-150 mM, between 100-150 mM, between 100-250 mM, between125-350 mM) can both (1) scavenge or interfere with radicals producedduring irradiation (e.g., e-beam or gamma) sterilization, and (2) notscavenge or interfere with radicals produced during UV inducedpolymerization. Viewed from a different perspective, tocopherol must bepresent in an amount that is effective to prevent runaway polymerizationwhen radicals are produced during e-beam or gamma sterilization, but noteffective to prevent polymerization in the presence of UV or othersuitable source of energy.

In some embodiments of the inventive subject matter, two types ofradical scavengers are included in a separator composition. A firstradical scavenger (e.g., E) can be sensitive to radicals that triggerpolymerization that are produced by ebeam or gamma irradiation. A secondradical scavenger (e.g., I) can be sensitive to radicals that triggerpolymerization that are produced by a photoinitiator. Therefore, whilenot wishing to be bound by any particular theory or limit the scope ofthe inventive subject matter, a technical effect of some aspects of theinventive subject matter is that E (e.g., tocopherol) can be used tomaintain a proper balance between A (photoinitator) and I (stabilizer)required for hardening of L (oligomer) in the presence of irradiationinduced formation of radicals. In other words, if there was an increasein I in an amount appropriate to scavenge radicals during irradiationsterilization (above 3 kG), the I in the composition would overwhelm A,and the composition would not cure upon exposure to a UV energy source.Compositions of the inventive subject matter could comprise I in a loweramount that allows for curing/hardening of the composition upon exposureto a UV energy source because of the presence of E. Viewed from anotherperspective, the E can be considered a sacrificial antioxidant that canbe dispensed with, and that does not interfere with a polymerizationreaction induced by A.

While not wishing to be bound by any particular theory, it is alsocontemplated that in some embodiments, a stabilizer I may not benecessary to allow for irradiation sterilization and separate UV curing.Experiments have shown that LAI does not maintain flowability whensterilized via irradiation. However, it is contemplated that an LAEcomposition can maintain flowability during irradiation sterilization toallow for subsequent UV curing when E is included in a concentrationthat is not effective to consume radicals generated by A, but effectiveto consume radicals generated during irradiation sterilization.

While the above data is based on L being L1, it should be appreciatedthat other oligomers (e.g., an acrylate containing oligomer and amethacrylate containing oligomer) are expected to work in place of Lbecause of their similar chemical makeup and use in free radicalpolymerization. Similarly, while the above data is based on A being A1(Additol BDK), other photoinitiators are expected to work in place of A1(e.g., a phosphine oxide photoinitiator, a ketone-based photoinitiator,and a benzoin ether photoinitiator) because of their ability todecompose into free radicals when exposed to light, and ability topromote polymerization reactions.

Additionally or alternatively, other stabilizers could be used in placeof phenothiazine (e.g., hydroquinone.) as they would be expected tosimilarly prolong storage and shelf life of the composition. Otherradical scavengers are also expected to work in place of tocopherol;however, other radical scavengers tested (e.g., BHA, BHT, carotene,ascorbic acid, bilirubin, gallic acid, and tempo nitroxide) were shownto interfere with some lab tests. Nonetheless, these scavengers can beused if the types of tests used are limited to specific clinicallaboratory tests. For example, certain antioxidants were found tointerfere with the measurement of some immunoassays but not withmolecular testing.

Based on the information provided herein, it is contemplated that aperson skilled in the art would be able to adjust radiation or otherparameters such that flowability of a separator substance havingdifferent constituents and concentrations thereof could be maintainedduring irradiation sterilization, and such that the substance couldsubsequently be UV cured. For example, the PHOSITA should appreciatethat a given composition is more likely to maintain flowability duringirradiation sterilization where the dose is delivered quickly (e.g.,e-beam vs. gamma). Additionally, the PHOSITA should appreciate thatincreasing antioxidant concentration (e.g., above 500 m) prolongs curetimes, and that increasing photoinitiator concentration to offset thiseffect (e.g., above 3%) adversely affects the function of theantioxidant in preserving the separator substance through sterilizationirradiation.

Unless the context dictates the contrary, all ranges set forth hereinshould be interpreted as being inclusive of their endpoints andopen-ended ranges should be interpreted to include only commerciallypractical values. Similarly, all lists of values should be considered asinclusive of intermediate values unless the context indicates thecontrary. All methods described herein can be performed in any suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g. “such as”) provided with respect to certain embodimentsherein is intended merely to better illuminate the invention and doesnot pose a limitation on the scope of the invention otherwise claimed.No language in the specification should be construed as indicating anynon-claimed element essential to the practice of the invention.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

What is claimed is:
 1. A sample collection tube, comprising: a tubehaving a lumen; and a separator substance disposed within the lumenhaving: a gel component layer material, wherein the gel component layermaterial comprises a thixotropic gel; and a non-thixotropic photocurablesealant component layer material that is distinct from the gel componentlayer material and comprises (1) an oligomer, (2) a photoinitiator, and(3) a stabilizer.
 2. The tube of claim 1, wherein the photocurablesealant component layer material polymerizes within 10 minutes to atleast 1 on the Shore 00 hardness scale when triggered by a suitableenergy source.
 3. The tube of any of claims 1-2, wherein the gelcomponent layer material has a density that is distinct from a densityof the photocurable sealant component layer material, and wherein eachof the gel component layer material and the photocurable sealantcomponent layer material has a density of between 1.00 and 1.09 g/cm³,inclusive.
 4. The tube of claim 1, wherein the separator substance has adensity between an average density of a cell-depleted fraction of wholeblood and a cell-enriched fraction of whole blood, and has a flowabilitysuch that the separator substance can settle under a centrifugal forceto a position between the cell-depleted phase of whole blood and thecell-enriched phase of whole blood.
 5. The tube of claim 1, wherein thephotocurable sealant component layer material is irradiationsterilizable without a loss of the flowability such that thephotocurable sealant component layer material can settle under acentrifugal force to a position between the cell-depleted phase of wholeblood and the cell-enriched phase of whole blood, and wherein thephotocurable sealant component layer material can be subsequentlypolymerized via UV curing.
 6. The tube of claim 1, wherein thephotocurable sealant component layer material comprises a promoter thatleads to polymerization within between 10-20 seconds to at least 1 onthe Shore 00 hardness scale when triggered by a suitable energy source.7. The tube of claim 1, wherein the photocurable sealant component layermaterial maintains at least one of a potassium level of the samplewithin 10% of an initial potassium level, and a glucose level of thesample within 5% of an initial glucose level for at least four days. 8.A method of producing a sample collection tube for blood separation,comprising: disposing a photocurable sealant component layer materialinto the lumen of a tube, wherein the photocurable sealant componentlayer material is non-thixotropic and comprises an oligomer, aphotoinitiator and a stabilizer; and subsequently to disposing thephotocurable sealant component layer, disposing a gel component layermaterial into the lumen of the tube, wherein the gel component layermaterial comprises a thixotropic gel; and wherein the photocurablesealant component layer material is polymerizable to a hardness of atleast 1 on the Shore 00 hardness scale upon exposure to a suitableenergy source for less than 10 minutes.
 9. The method of claim 8,further comprising disposing a sample into the lumen of the tube, andcentrifuging the sample collection tube with the sample, the gelseparator component layer material, and the photocurable sealantcomponent layer material.
 10. The method of any of claims 8-9, furthercomprising sterilizing the sample collection tube with the gel separatorcomponent layer material and the photocurable sealant component layermaterial disposed therein via irradiation sterilization, whilemaintaining a flowability of the photocurable sealant component layermaterial with whole blood.
 11. The method of claim 8, wherein thephotocurable sealant component layer material comprises a promoter thatenables it to polymerize to the hardness of at least 1 on the Shore 00hardness scale upon exposure to the suitable energy source for less than10 minutes.
 12. The method of claim 8, wherein the photocurable sealantcomponent layer material and the gel separator component layer materialeach have a density between an average density of a serum fraction ofwhole blood and a cell-containing fraction of whole blood, and isfurther flowable in whole blood.
 13. The method of claim 8, furthercomprising heating the sample collection tube to at least 250 degreesCelsius to sterilize the sample collection tube while maintaining aflowability of the photocurable sealant component layer material withwhole blood.
 14. The tube of claim 1, wherein no more than 25% of theseparator substances comprises the non-thixotropic photocurable sealantcomponent layer material.
 15. The tube of claim 14, wherein no more than10% of the separator substances comprises the non-thixotropicphotocurable sealant component layer material.